Day 16

Ink Microinjections
In order to determine whether the microinjections given to the rats throughout the experiment were isolated to both sides of the parabrachial nucleus, we give rats microinjections of dye.  Whoever is doing histology will then cut the brain into pieces, put them on slides, and examine them under a microscope to see where the dye is located.  Our goal is for the dye to only be in the PBN (bilateral).  Lindy let me give the rat the ink microinjections.  First, they are injected with anesthesia.. Lindy let me wrap the rat for its injection as well, which was a lot more difficult than I expected.  Every time I correctly positioned the rat, he would move.  Sometimes he would even grab the cloth and unfold it when I picked him back up to try again.  The time I finally got him wrapped properly, it was not tight enough and he could still move when the needle was being injected.  Lindy eventually had to go ahead and do it herself.  We then waited for the rat to go unconscious.  I unscrewed its dummies and placed the injectors in the cannulae.  I also set up for microinjections.  The hardest part was getting the tubing onto the microinjector without having air bubbles.  There would be air bubbles where the microinjector inserted into the tube, and  I poked a hole in the tubing a couple times.  It took a few tries to finally get it right.  Once the microinjections were complete, I would hand the rat with its label to Dr. Holstein for perfusions.

Cleaning
Julia and I deep cleaned the testing room.  We took apart the testing boxes and wiped down all the solution that had stuck to the counters and floor.  This is important so the mess does not accumulate over time. 

Day 15

CTA Testing
Today, we began CTA testing with group C and group D.  Before testing them, we had to weigh all the rats.  We could run a maximum of 8 rats at a time.  We first gave each rat saccharine for one hour and then injected them with LiCl directly after.  The LiCl makes the rats feel sick which they then associate with the saccharine they just drank.  The goal is for their lick count to decrease the next day when they are given the saccharine again.  We run each rat and then clean out their cages in between.  After the rats finished with CTA testing, we reattempted microinjections for three rats.  The air bubble only moved in both tubes for one rat so we ran him alone and quit trying the reinjections for the others.  We agitated the cannulae of all other rats.

Day 14

Microinjections

Today, we did microinjections for the last time with group C and group D.  We began with group D1 and had to reinject both rats receiving CDP for the air bubble to move.  All rats today received 0.05M quinine.  While group D1 rats were running, we gave water to group C for 60 minutes.  The rats in group D1 had very few licks, which Dr. Holstein attributed to quinine.  While group D2 was running, I made and labeled jars with subject numbers for group C and group D.  For group D3, we had trouble, again, with the CDP in which both rats had to have more than one microinjection.  Group C and group D will continue with CTA tests.

Day 13

Microinjections

The first set of microinjections went well for group C1.  However, the second rat receiving CDP had problems.  He received three microinjections on the right side, and the air bubble still didn’t move in the tubing.  We decided to run him today anyways and retest him another day as well.  We also had trouble with the rats in group C2 receiving CDP.  The first rat had three microinjections, but thankfully the second rat only needed one microinjection for the air bubble to move.  All rats in group C1 and group C2 received 0.01M quinine.  Group D rats were given 60 minutes of water.  While group C2 was testing, I prepared jars with formalin.  I labeled 17 jars total with the subject number of each rat in group A and group B.  I then filled the jars about 2/3 full with formalin.  We ran group C3 rats, and only had to reinject the first rat receiving CDP once before the air bubble moved.  Rats in Box 1, Box 2, and Box 3 received 0.01M quinine while the rat in Box 4 received 0.03M NaCl.  One rat only had one lick.  Some rats in group A and group B were retested for CTA.  The other rats of group A and group B who successfully experienced taste aversions the previous day were finished testing.  I was able to see the preparation for tissue sampling performed by Dr. Holstein and Dr. Pittman.

Day 12

Microinjections

We ran group D rats today, starting with group D2.  The first two rats’ microinjection ran well, but the second rat receiving CDP had to have a second microinjection on the left side.  When I inserted his left dummy back in his cannula, a little liquid was coming out.  All rats in group D received 0.01M quinine.  I gave 60 minutes of water to group C while group D2 was running.  For group D3, all aCSF microinjections went well.  The first rat receiving CDP had to have two microinjections, and the second rat receiving CDP had to have three microinjections.  I changed out the bedding for the D2 rats while they were running and took out the trash with Alex.  For group D1, all aCSF microinjections went well, and both rats receiving CDP had to have two microinjections on the left side for the air bubble to move in the left tubing. 

CTA Testing

Julia and I prepared four syringes with LiCl to use for any rats that not react aversely to the NaCl.  They would need to be reinjected with the LiCl after drinking the NaCl to establish an aversive relationship.  We encountered some problems when preparing the syringes.  Some needles did not securely fit on the syringes, and we had more trouble today than yesterday getting rid of the air bubbles in the LiCl solution.  It took longer, but we finally managed to properly prepare the four syringes.

Day 11

Microinjections

Today, we are testing rats in group C.  This morning, I came in and prepared the syringes for microinjections.  I cleaned the fragile microinjectors with ethanol and distilled water.  I then filled them with water and attached them to the pump.  I also prepared the tubes for microinjections.  After I vortexed the drugs, we began microinjections for group C2.  All rats received 0.01M quinine (bitter).  All aCSF injections went well.  The first rat receiving CDP had problems with the left side.  It took three microinjections for the air bubble to move on the left side.  The second rat also had trouble, and when I reinserted his dummy into his right cannula, liquid was coming out.  Group C3 rats all received 0.01M quinine except for the rat in Box 4 who received 0.03M NaCl.  Group C1 rats all received 0.01M quinine.  We gave water to group D rats for 60 minutes, and agitated the cannulae of and weighted rats in group A and group B.  I discovered that rats can unscrew their dummy if it is not properly secured.  When agitating one cannula of a rat, his was missing for the second time because he had methodically taken it out.

CTA Testing

Dr. Holstein taught us how to prepare syringes for CTA (conditioned taste aversion) testing.  First, you take the barrel and move the plunger in and out a few times in the LiCl to get rid of air bubbles. Then you hit the barrel against your hand to move the air bubbles to the top.  After they are at the surface, you pull back on the plunger so there is air in the barrel and push the plunger back in until liquid comes out.  You can then screw on the needle tightly, pop off the cap, and squirt the liquid out of the needle.  We prepared 16 syringes with LiCl to inject group A and group B rats with.  The LiCl was injected to the left or right of the midline about an inch above the base of the tail.  It is important to pull back on the plunger when injecting the rats to make sure no blood is coming out.  LiCl will make the rats feel sick, which we hope they will associate with the NaCl they are given to drink (rats in group A and group B had not been given NaCl before, so it is a new taste for them).  Julia and I did not yet experience CTA testing, but will have that opportunity when it is time for the rats in group C and group D.

Day 10

Microinjections

Today was a much simpler day, but we encountered a lot more problems.  Group A and group B are done with microinjections until CTA testing.  We gave microinjections only to group C today.  Of group C3, rats in Box 1 and Box 2 received 0.3M NaCl, and rats in Box 3 and Box 4 received 0.5M NaCl.  Group C1 rats received 0.05M saccharine, and group C2 rats received 0.3M MSG.  For group C3, all aCSF injections went well.  However, the first rat receiving CDP had to be reinjected on both sides.  The second rat receiving CDP was reinjected on the left side twice for the air bubble to move in the left tubing.  Two rats from group C1, one receiving aCSF and one receiving CDP, had to be reinjected for the air bubble to move.  We were behind schedule today due to the many difficulties with the microinjections.  Julia and I agitated the cannulae of the rats in group A and group B.  We also gave the rats in group D water for one hour.

Day 9

Microinjections

Today, we continued microinjections for group C.  The first round of microinjections went well for group C3, and they were all given .01M saccharine. We gave water to group D for 60 minutes while group C3 was testing.  A problem we encountered a few times was that the air bubble went down, but not to the point where the correct amount of CDP was injected.  In this situation, we cannot give them another dose of the drug, so we have to test them with what was actually injected.  Group C1 rats in Box 1, Box 2, and Box 3 received 0.1M MSG, and the rat in Box 4 received .01M saccharine.  The air bubble in the left tube of one rat in group C1 and one rat in group C3 did not move, so the left side was reinjected.  When the injector was taken out of the left side for the rat in group C2, it began to bleed a little so we agitated his cannula after he tested.   All rats in group C2 received .1M MSG.  All other injections were good.  Only one rat did not have many licks.  Group C was given water for 15 minutes after all of them had been tested, and we agitated the group D rats’ cannulae.

Day 8

Microinjections

This morning, we ran group D rats.  All of group D1 received 0.3M Saccharin, all of group D2 received 0.3M MSG, and rats of group D3 in Box 1 and Box 2 received 0.3M NaCl and rats of group D3 in Box 3 and Box 4 received 0.5M NaCl.   We had another issue with the microinjections.  During microinjections for the D2 set of rats, the aCSF injectors seemed to be clogged because they did not run properly.  This put us back a little on time, but we quickly made up for it.  A rat in the D3 group had to have a second microinjection on its right side because the air bubble did not move on the first try.  While the rats were running, Julia and I made the solutions for the next trials.  We added 400 mL of water to the MSG concentration and allocated them equally (about 100 mL each) between the four baby bottles.  We also added 200 mL of water to each of the NaCl concentrations and then allocated them between the four baby bottles. After adding the water and before pouring the solution in the bottles, we added a stir bar to the beaker and allowed the solution to stir for 10 minutes.  We covered the baby bottles with cling wrap until they were ready for the next trial.  We also gave group C water for 60 minutes and group D water for 15 minutes.

Day 7

Microinjections

Today was a CRAZY day! We ran a total of five groups.  I helped run three of them, all of which were in group C.  All rats in group C1 and all but one rat in group C2 received 0.1M MSG (salty), and all rats in group C3 and one rat in group C2 received .01M saccharine (bitter).  Because this was the first round of microinjections for this group of rats, their left cannula needed to be painted with the purple nail polish.  The rats in group C squirmed more during injections than the rats in group A and group B, but that may have been because it was their first time undergoing microinjections.  It took two tries for the air bubbles to move in both tubes and the CDP to be injected for one rat, and it took two tries for the CDP to be injected into the right side of another rat.  Both of these rats seemed to have a clogged cannula.  All other injections were good.  We agitated the cannulae of group D rats so they will be ready for microinjections tomorrow.  The dummy in the left cannula for two different rats in group D was very tight and needed to be cleaned.  It was difficult to put the dummy back into the cannula, so we took it out and cleaned it because we were scared it would break in the rat’s cannula if we tried to put it back in.  Microinjections continued for group B this afternoon.

Day 6

Microinjections

Today was another day of microinjections.  Group A was tested.  They received the same drug as they did the first time they were tested and are placed in the same box, however, Box 1 and Box 2 received the .003M citric acid and Box 3 and Box 4 received the .01M sucrose.  I also vortexed the drugs before they were used to make sure the solution was completely mixed.  Group B was given water for 60 minutes and their cannulae were agitated.  For one rat, we had the same problem that we did the other day.  It took three tries until the air bubble finally moved for CDP in the right side.  The drug was delivered using the left tubing.  We also had to replace a few injectors because the rats moved while they were being put in, causing them to bend or break.  At the end of the day, we cleaned the cages, gave group A 15 minutes of water, and relabeled the baby bottles with numbers one through four.

Day 5

Microinjections

We did more microinjections today on Group A again.  Each rat was placed in the same Box in the testing room as they were placed in before, but this time the rats received the opposite treatment (those with CDP last time were given aCSF, and those with aCSF last time were given CDP).  We continued to agitate the cannulae of the rats that were not being tested.  One problem encountered during microinjections was that the air bubble did not move in the tube on the right side.  When the air bubble doesn’t move, it means the drug is not being injected.  The injection was attempted again using the left tubing, but the air bubble still did not move.  The injector was cleaned and reinserted into the tube.  This time the microinjection was successful (the problem was most likely due to a clogged injector).  This put us back a little on time, but everything else ran smoothly.  We ended the day again by giving 15 minutes of water to the rats.

Day 4

Microinjections

Today we continued doing microinjections.  We tested group B using the same procedures as we did for group A yesterday.  It was a very successful day with all microinjections going well.  The only difficulty we experienced was getting rid of all the air bubbles in the tubes.  The tubes that were used for the aCSF injections continued to have air bubbles and had to be prepped multiple times before it could be used for injections.  Also, one of the injectors broke and needed to be replaced before microinjections could continue.  In the testing room, Box 1 and Box 2 are given sucrose (sweet) and Box 3 and Box 4 are given citric acid (sour).  A different concentration is given to each group creating a concentration curve.  Box 1 and Box 3 contain the rats injected with aCSF and Box 2 and Box 4 contain the rats injected with CDP.  This is the same as yesterday.  There are still a large number of rats that need surgery by Sunday.

Tomorrow: We will continue doing microinjections.  I will get to practice delivering the microinjections to a rat whose cannulae were not correctly placed in the PBN during surgery.  We cannot use this rat for research purposes (we are testing injections strictly in the PBN), and he will be replaced by a backup rat.

Day 3

Microinjections

Today we learned how to prepare and deliver microinjections to the rats’ PBN.  There is a very elaborate process that goes into prepping.  First, the microinjectors are sterilized and flushed.  They are very fragile and expensive, so it is important to be careful.  The plunger must be pulled out slowly and should not exceed the 1.0μL mark (0.9 μL is a good amount).  The microinjector is filled with ethanol and then injects the ethanol into the waste beaker 3-5 times.  It is then filled with water and injects the water into the waste beaker 6-10 times.  This ensures that there is not leftover ethanol injecting into the rats’ brains.  The microinjectors are inserted comfortably into the injector machines (the right and left microinjector should be inserted into the proper side).  A syringe is then filled with water and releases the water into the tube several times.  There should be no air bubbles.  The syringe is then removed, and the microinjector is inserted into the tube.  Again, there should be no air bubbles.  Clay can be used to hold the tubes when they are not in use.  When one of the two drugs (treatment: CDP control: aCSF) is ready to be injected into the rat’s PBN, an air bubble is formed in the tube before the specified drug is sucked up into the tube of both the right and left side.  During this time, I have removed the rat’s dummies and will paint the left dummy tip with nail polish to differentiate it from the right.  The injectors on the tip of the tubes are inserted into the correct cannula.  The injector machine inserts 0.2μL of the drug into the rat’s PBN per minute.  After the drug has been injected into the PBN, we wait two minutes while the injector sits into the cannula.  Then we remove the injector and screw back in the dummies.  The rats are placed in an assigned cage in the testing room, are given a specific flavor of liquid to drink, and their licks are recorded in a 60 minute time period.  We also replaced their cages with a new one for when they complete the experiment.  We tested the water restricted rats using an ABBA within subjects design.  The tubes were cleaned again after the second microinjection and then at the end of the experiment.  The cages in the testing room were wiped using an antibacterial wipe and dried using a paper towel after each trial.  Notes are made for each rat.

Difficulties

There were three difficulties.  One was having enough skill and good eyesight to place the dummies back into the cannulae of squirming rats.  The second was that one rat did not lick the baby bottle at all.  This was one of the same rats that did not lick the bottle during training.  A third difficulty was that one rat had scratched and irritated his head, so he had to be run in a separate trial by himself after he was cleaned up.

Day 2

Today, I practiced handling the rats.  I scratched the rats' heads that did not yet have surgery to get them used to their heads being touched.  For the ones that had surgery, I practiced removing their dummies and placing them back into their cannulas.  If the rats were still, I put the dummy back in on the first try.  If the rats squirmed, it took much longer.  One rat in particular moved a great deal, and I had to get Julia's help to place the dummy back into the cannula.

Day 1

Experiment Training 
Today was a very eventful day with much to learn.  I first learned how to fill the baby bottles with 0.2 moles of sucrose (about 3/4 full), place the stopper on, and secure it with a rubber band ensuring that the small white rubber disk is not covered so it can properly sit in the holder.  Every morning, I have to log onto the computers so they are ready to run.  We had a set time to "cuddle" with the rats and pet the heads of the ones who have not yet had surgery.  This gets them used to their heads being touched.  For the ones who have had surgery, we were able to practice taking out the dummies and placing them back into the cannulas (it is important not to mix up the right and left dummy).  There are two ways to do this: one way is to hold the rats on your chest where the pocket is and the second is to hold the rat in the crook of your arm.  I found the latter to be best for me because it is easier to see the hole in order to place the dummy back in the cannula.  Today, we ran the rats who have not had surgery in a training session.  This gets them used to the equipment and being transported, so they are not intimidated during the actual test.  Each rat is placed in a separate clear, plastic cage with the sucrose water.  The lights are turned off with only a red lamp to see.  A device records the number of licks by each rat in a 60 minute period.  Rats that had a low number of licks during the time period were rerun.  The bottle weights are recorded before and after each trial.  After the rats are placed back in their original cages, the cages used for the trials are wiped down with sterile wipes and dried.  At the end of the day after all test runs, the computer and lights are turned off and the bottles cleaned.

Surgery
I was also able to watch Dr. Pittman perform a surgery today on a rat.  The rat is first anesthetized with two injections, one to the right and one to the left of the midline about an inch above the base of the tail.  When injecting, it is important to pull up on the plunger and make sure there is no blood before pushing down.  Once the rat is asleep, it is secured in a stereotactic apparatus.  Once everything is in the correct place, it is time to start the surgery.  The skin is cut using a scalpel and the connective tissue is pulled back.  The most tedious part of surgery is to ensure that the rat is in a particular position so the cannula is placed in the correct location.  This should not be rushed because it would be a flawed experiment if the microinjections were not delivered to the parabrachial nucleus (PBN).  The rat's lambda and bregma are used to make sure the rat is correctly positioned.  The distance has to be measured each time the head is moved.  It is important also to pay attention to when the coordinates need to be zeroed.    Based on previous research, the stereotaxic atlas is used to locate the PBN.  Circles are drawn in sharpie.  Small holes are drilled into the rat's skull where screws are inserted as an anchor for the cement.  Dr. Pittman allowed me to drill one of the holes in the skull which was very interesting.  After four screws are fastened, the cannula is inserted into either the left or right hole.  A gel pad is placed over the other hole while the cement secures the cannula in place and hardens (this is a precaution so the other hole does not fill with cement).  Once the cement hardens, the same procedure is repeated on the other side.  During the entire surgery, it is crucial to sterilize all tools and to recheck measurements multiple times.  It can also help to use a microscope.

Tomorrow: I will go into the lab and practice handling the rats and placing the dummies into the cannulas.